1.2 Protocol for Adding GFP into Vesicles
- 100ml glass beaker x1
- Gilsson pipette (200µl) + pipette tips
- Nitrogen tap
- 1000µl pipette tip
- Desiccator connected to a vacuum
- 25ml Glass pipette x1
- Sonicator with medium-sized probe
- 25°C incubator
- 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) 20mg/ml in chloroform, ≥99.0%
- Mineral oil
(Note: Alternatively, use POPC and dodecane)
Preparing the lipid-oil suspension for the inner leaflet
- Place 125 µl of the 20 mg/ml DOPC solution in a 100-ml glass beaker. (Equipment should generally be made of glass and not plastic so as to prevent adhesion of lipid molecules to plastic surface)
- Using plastic tubing and a 1ml pipette tip, evaporate the chloroform under nitrogen to obtain a dry, thin lipid film. (Rubber tubes are not recommended as they are more likely to emit debis into the lipid film)
- Put the beaker in a desiccator connected to a vacuum for 1hr. (This is to remove the chloroform)
- Add 50 ml of mineral oil to reach a final lipid concentration of 0.05 mg/ml
- Place the beaker containing the suspension in the ice bath
- Sonicate suspension for 30 min (Pulse 1, ~10 Amp). (This is to disperse the phospholipids)
- Leave overnight at 25°C to ensure that the lipid molecules are fully dispersed in oil
- Magnetic stirrer
- Aluminium foil
- 200µl pipette + tips
- 1000µl pipette + tips
- 50ml 1-inch diameter glass centrifuge tube
- 1-inch diameter tabletop centrifuge
- 5ml syringe + long 16-gauge stainless steel needle x1
- Test tube x1
- Solution A
- Tris buffer
- GFP standard Solution
Preparation of Solution A
- Prepare a 10ml solution A with 100 mM NaCl and 5 mM Tris buffer at pH 7.4
Emulsifying the Aqueous Solution
- Separate about 5 ml of the lipid-oil suspension into a glass container. (For the interface preparation)
- Prepare 10X GFP standard solution with Solution A to form 1ml aqeous solution.
- Add 250 µl of the aqeous solution to the 45ml lipid-oil suspension in mineral oil.
- Wrap the beaker in aluminium foil. (This is to prevent bleaching of GFP)
- Gently stir the mixture with a magnetic stir bar for 3 hours.
Preparing the interface (While emulsion is mixed)
- Place 2 ml of lipid-oil suspension over 3 ml of solution A in a 1-inch-diameter centrifuge tube.
- Leave for 2–3 h for lipids to achieve the coverage of the interface surface (>3h and the lipid may start to clump together)
(Note: This step can been modified to use 2ml of emulsified solution instead of the lipid-oil suspension)
Formation of bi-layer vesicles
- Pour 100 µl of the inverted emulsion over the interface. (Note: This step is omitted if 2ml of emulsified solution is used instead of the lipid-oil suspension)
- Centrifuge at 120 x g for 10 min
(Note: Alternatively, centrifuge at 30 x g for 20 min)
Collecting the vesicles:
- Using a 5-ml syringe with a long 16-gauge stainless steel needle, collect some of solution A.
- Expel some of the solution to remove all air from the syringe and needle. (Expelling most of the Solution A would ensure a less diluted solution of vesicles)
- With the tip of the needle in the aqueous phase, gently expel the solution contained in the syringe. (This prevents the extraction of the lipid-oil suspension when the needle is plunged into the tube)
- Gently recirculate the buffer several times.
- Aspirate most of the solution into the syringe, and remove the needle from the solution. (Be careful not to aspirate the lipid-oil suspension)
- Wipe the tip of the needle clean.
- Unload the vesicle suspension into a test tube and store in the dark.
- Use optical microscopy to check that the vesicles obtained were not deformed or aggregated. Ideally, the protocol should yield ~109 vesicles of 1µm diameter. (Caution: Over-exposure of light under the microscope would bleach the GFP!!)
- Time required for Day 1: ~ 2h; for Day 2: ~4h.
- The protocol is based on Engineering Asymmetric Vesicles by Sophie Pautot, Barbara J. Frisken, and D. A. Weitz.
- Modifications to protocol:
- The original protocol uses anhydrous 99:1 dodecane:silicone oil solution instead of mineral oil
- The original protocol uses POPC instead of DOPC phospholipids
- The original protocol sonicates the suspension in a cleaning sonic bath for 30 min
- Use of GFP as a visual signal may require osmolarity considerations